Chk2 is a serine/threonine protein kinase found mutated in certain hereditary and sporadic cancers. Ionizing radiation (IR) activates the kinase activity of Chk2 in a phosphorylation-dependent manner. ATM phosphorylates Chk2 on threonine 68, which promotes oligomerization and phosphorylation on threonines 383 and 387 within the activation loop of the catalytic domain. In this study, threonines 68, 383, and 387 were confirmed as sites of Chk2 phosphorylation both in vitro and in vivo. In addition, serine 516 was identified as a novel IR-inducible phosphorylation site in vivo and as a site of autophosphorylation in vitro. Interestingly, Chk2 was capable of autoactivation in the absence of IR when overproduced in bacteria, in 293 cells, and in murine embryonic fibroblasts lacking Chk2. A kinase-inactive mutant of Chk2 was phosphorylated on T68 and T383/T387 but not on S516 in cells containing Chk2 and on T68 but not T383/T387 or S516 in cells lacking Chk2. This establishes a dependency on Chk2 kinase activity for phosphorylation of T383/T387 and S516 but not for T68 in vivo. We demonstrate that T68 phosphorylation is regulated by kinases in addition to ATM and Chk2. Taken together, our data indicate that autophosphorylation of Chk2 can occur both in cis and in trans and suggest that oligomerization may regulate Chk2 activation by promoting these cis- and trans-phosphorylation events. The importance of oligomerization is underscored by the observation that substitution of isoleucine for threonine at position 157, a mutation found in a subset of patients with Li-Fraumeni syndrome, impairs both Chk2 oligomerization and autophosphorylation.